The endoplasmic reticulum (ER) is a specialized folding environment in which nearly one-third of the proteins encoded by a eukaryotic genome are translocated and folded as either luminal secreted proteins or transmembrane proteins. Proteins are exported from the ER by the concatamer complex II (COPII) machinery which generates transport vesicles for delivery of cargo to the Golgi (Lee et al., Annu. Rev Cell Dev. Biol. 20, 87 (2004)). The ER-associated folding (ERAF) pathways are also coordinated with ER-associated degradation (ERAD) pathways whereby misfolded proteins are targeted for translocation to the cytosolic proteasome system (Wegele et al., Rev Physiol Biochem Pharmacol 151, 1 (2004); Young et al., Trends Biochem. Sci. 28, 541 (2003)). For cytosolic proteins, there is also protein folding quality control system involving molecular chaperones and the ubiquitin-proteosomal and autophagic degradation pathways (Kubota, J. Biochem. (2009)).
Numerous misfolding diseases occur when normally folded protein expressed on the cell surface, extracellular environment or the cytosol misfolds due to pathologic conditions, resulting in misfolded aggregates. Neurodegenerative diseases, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease/Lewy body disease (PD, LBD) are associated with neural deposits of misfolded aggregates of normal protein, including tau and the Aβ fragment of the amyloid precursor protein (APP) in AD; superoxide dismutase-1 (SOD1) in ALS; and α-synuclein in PD and LBD. Misfolding of the cellular prion protein, a cell surface glycoprotein expressed in nervous system and other tissues, is implicated in Creutzfeldt-Jakob disease (CJD) of sporadic and familial origin, Gerstmann-Straussler-Scheinker syndrome (GSS), kuru, and fatal familial insomnia (FFI). The normally folded prion protein (denoted PrPC) can adopt a pathogenic misfolded conformation induced by physical contact with the disease-misfolded prion protein (generically denoted PrPSc) in a process designated template-directed misfolding. The tertiary and secondary structure of PrPSc differs substantially from that of PrPC leading to solvent exposure of previously buried residues that in this invention are used as the basis for distinguishing one form from the other. The original aggregate of misfolded prion protein that seeds the misfolding process can either arise endogenously, as in sporadic CJD, or be acquired as an infectious agent from another animal or human, as in variant and iatrogenic CJD. In the former case, certain inherited mutations of the Prion Protein gene (PRNP) coding sequence can predispose individuals to misfolding of PrPC and trigger clinical manifestations of the disease. At present, there is no curative therapy for prion disease, resulting in a mortality rate of 100% (Prusiner, Ann Rev Microbiol 48, 655 (1994)). Prion protein is implicated in animal diseases as well. Those of economic interest include bovine spongiform encephalopathy (BSE) of cattle, scrapie of sheep, and chronic wasting disease of cervids. As with human disease, there is no effective treatment or vaccine prevention for animal prion diseases.
Protein misfolding diseases are not limited to those of the central nervous system. The misfolding and aggregation of the thyroid hormone carrier protein transthyretin (TTR) is involved in familial amyloid polyneuropathy and senile systemic amyloidosis (Benson and Kincaid, Muscle Nerve 36 (4), 411 (2007)). Patients on protracted hemodialysis for kidney failure may suffer from renal accumulation of β2 microglobulin amyloid deposits, a component of the type 1 major histocompatibility complex (MHC) present on most cells of the body (Winchester et al, Adv Ren Replace Ther 10(4), 279 (2003)).
Diseases of protein aggregation can be considered in two broad classes: those involving natively unstructured proteins or peptides (e.g., alpha synuclein and Aβ respectively), and those involving proteins possessing structured domains (e.g., PrPC and TTR). According to one hypothesis, natively structured proteins are thought to have been evolutionarily selected to remain soluble in physiological conditions; i.e., they do not spontaneously aggregate.
Protein misfolding also occurs in cancerous cells where a generalized decline in folding fidelity results in misfolded protein residing at the cell surface. In cancer cells, dysregulation of cell cycle control results in their uncontrolled proliferation. Left unchecked, the growth and dispersion of these cells through metastasis can cause anatomic and functional derangement of organs and compete for vital nutrients. A normal human cell is subject to careful regulatory control by many signalling mechanisms to prevent division except when needed for tissue repair or renewal. Cancerous cells accumulate somatic mutations that render them insensitive to these signals. A consequence of these and other mutations is a decline in protein folding quality control: a healthy cell is able to recognize and destroy synthesized protein that has not correctly folded, but some cancer cells display reduced fidelity and monitoring of folding which can lead to incorporation of misfolded proteins into the plasma membrane (reviewed in Nature Vol 426 No 6968 pp 883-909). The ectodomains of these misfolded proteins expose to the extracellular environment protein surfaces that are normally buried in the molecular interior, and as such present epitopes for selective identification. Thus misfolded cell surface proteins can provide a diagnostic tool and therapeutic targeting for discrimination between cancerous cells and normal cells that retain their correct protein folding machinery.
Folding fidelity is expected to be particularly impaired with the overexpression of certain proteins by selected tumor cells Which membrane proteins are over-expressed depends on the form of cancer. For example, some aggressive breast cancers over-express HER2 (Milanezi et al, Expert Rev Mol Diagn 8 (4), 417 (2008)), while some lung and colon cancers overexpress EGFR (Ciardiello and Tortora, N Engl J Med 358 (11), 1160 (2008)). CD20, a membrane-spanning protein of unclear function, is expressed on the surface of B-cell lymphomas, hairy cell leukemias, and B-cell chronic lymphocytic leukemias. CD38 is associated with leukemias and myelomas, while CD44 is a surface glycoprotein implicated in colon cancer metastasis. Targeting these various receptors provides a mechanism for identifying cancerous cells and directing cytotoxic treatments toward them.
Activating pro-apoptotic signalling pathways on cancerous cells is a related way of promoting eradication of the cancer. The Fas receptor (FasR) is involved in triggering apoptosis through an intracellular signalling cascade, so an agonist binding to FasR can trigger death of the cell (Daniel and Wilson, Curr Cancer Drug Targets 8 (2), 124 (2008)). One approach of exploiting this mechanism as a therapeutic strategy may be to selectively activate Fas receptors on cancerous cells, as nonspecific Fas activation generally leads to apoptosis of healthy cells expressing the Fas receptor. The Notch signalling pathway is involved in cell fate decisions, and dysregulated expression of the cell-surface Notch receptor, ligands and targets has been implicated in cervical, head and neck, endometrial, renal, lung, and breast carcinomas, pleural mesotheliomas, and malignant melanomas, as well as Hodgkin lymphomas, anaplastic large-cell non-Hodgkin lymphomas, and some acute myeloid luekemias and B-cell chronic lymphoid leukemias (Nickoloff et al, Oncogene 22, 6598 (2003). Blocking Notch signalling on cancerous cells by interference with the extracellular domain of Notch protein offers a way to attenuate the stimulus required for continued uncontrolled proliferation. Ideally, this interference should be limited to cancerous cells, as other non-malignant cells employ Notch signalling. Specifically targeting misfolded Notch receptor would accomplish this, as healthy cells will not present misfolded Notch to the extracellular environment. Additionally, the presence of misfolded Notch receptor protein on the cell surface can be used as an indicator of malignancy, enabling induction of an immune response against malignant cells expressing misfolded Notch by administration of an antibody against Notch specific for the misfolded conformation.
The overexpression and impaired folding fidelity expected in certain cancer cells is likely accompanied by partial unfolding of protein structured domains.
The protein unfolding process can be explained by studying free energy changes in proteins. The Gibbs free energy is the thermodynamic quantity minimized for spontaneous reactions occurring at constant pressure (Atkins and de Paula, Physical Chemistry, 7th Edition (2002)). Its change during a chemical process ΔG is a function of a reaction enthalpy change ΔH, the reaction entropy change ΔS, and the reaction temperature T:ΔG=ΔH−TΔS 
Alternatively, the Helmholtz free energy change ΔF=ΔU−TΔS, which replaces the enthalpy change with the internal energy change ΔU, is nearly equivalent since protein unfolding involves a small change in the volume of the protein system and may also be calculated. For the process of protein unfolding, ΔH and ΔS are complicated functions that depend on many parameters of the protein's structure, including the hydrogen bonding network, topology of folding, interactions with solvent, and presence of post-translational modifications. The Gibbs free energy change is related to the equilibrium occupation of the folded and unfolded states by the equilibrium unfolding constant K:
  K  =                              [          Protein          ]                unfolded                              [          Protein          ]                folded              =          exp      ⁡              (                  -                                    Δ              ⁢                                                          ⁢              G                        RT                          )            
If the free energy of occupation for all partially unfolded states of a protein is known, the partition function Z may be constructed as a sum of Boltzmann factors over all partially unfolded states:
  Z  =      ∑          exp      ⁡              (                  -                                    Δ              ⁢                                                          ⁢              G                        RT                          )            
The equilibrium probability of occupation for a given partially unfolded state i is then:
      P    i    =            exp      ⁡              (                  -                                    Δ              ⁢                                                          ⁢                              G                i                                      RT                          )              Z  
The difference in conformation between normally folded and misfolded protein introduces the concept of a disease-specific epitope—a region of the protein that is uniquely solvent exposed in the misfolded form. Antibodies raised against this epitope will therefore have the ability to bind exclusively to the misfolded protein, which is of use for diagnosis by identifying the presence of the misfolded protein in patient specimens. The application of antibodies against disease specific epitopes for treatment depends on the circumstances of the disease: in the case of cancer cells presenting misfolded protein, they can mark the cancerous cells for destruction by the immune system; for amyloidoses, they work by inhibiting the recruitment of additional misfolded monomers to the amyloid fibril, by marking the misfolded proteins for immune-mediated phagocytosis and destruction, and perhaps by also preventing template-directed misfolding by blocking binding of misfolded protein to normally folded protein.
In order to identify useful disease specific epitopes, a high resolution structure of the misfolded protein aggregate may provide a rational starting point for prediction. Unfortunately, technical limitations have rendered futile such atomic level structures for PrPSc, amyloids composed of Abeta, TTR, or Thy-1, or any misfolded protein on cancer cells, obviating predication of any stable discontinuous epitope or structured conformational epitope. A further limitation of this approach is that misfolded proteins may exist in an ensemble of interchangeable forms, with stable conformational epitope formation likely being non-universal. It is also in principle possible to identify misfolding specific linear epitopes experimentally by an exhaustive method comprising raising antibodies against all possible epitopes contained within the protein and screening them for selective reactivity toward the misfolded forms. However this “shotgun approach” for the thousands of overlapping linear epitopes in a medium-sized protein is likely to be expensive, laborious, and time-consuming. It is therefore desirable to rapidly and rationally limit candidate epitopes to ten or fewer per protein that can then be rigorously characterized for suitability as therapeutic or diagnostic targets.
From a diagnostic and therapeutic point of view it is important to identify disease-relevant epitopes caused by protein misfolding. Current strategies have focused on preparing misfolding-specific antibodies using whole misfolded protein as an immunogen. This approach has several disadvantages. Recombinant misfolded protein may not expose the disease-relevant epitopes due to differences between the in vitro and in vivo misfolding mechanisms. Furthermore, misfolding-specific epitopes are poorly recognized in the context of whole proteins (like PrPSc), and whole protein immunization runs the risk of immune recognition of native surface epitopes that could trigger autoimmunity.